The proposed project will explore the roles of pleiotropy and epistasis in adaptive evolution and integrate ideas and questions from biophysics and biochemistry into an evolutionary framework in a bacteriophage system. The experiments, inspired by the population dynamics experienced by many pathogens, will use a unique experimental-evolution protocol involving rapidly fluctuating selective pressures to induce a two-component fitness based on growth rate and one of three biophysical parameters: capsid stability, low-pH tolerance, and novel host binding. The selection protocol consists of periods of growth within hosts punctuated by strong selection for one of three biophysical properties in the absence phage replication. For Aim 1, this protocol will be used to study the pleiotropic effects of individual beneficial mutations in five microvirid bacteriophage genotypes on growth rate and the three biophysical properties and to determine how this conflict, and pleiotropy in general, affects the genetic variation available for adaptatio. For Aim 2, beneficial mutations identified for Aim 1 will be engineered into new genetic contexts to reveal the extent to which biophysical properties of mutations are additive across backgrounds. The results from Aim 3 will be used to determine whether long-term adaptation can allow deleterious pleiotropic effects to be overcome through compensatory evolution to allow the two traits to be simultaneously maximized. The results from the proposed project will serve as a bridge between biophysics and evolution by subsuming biophysical parameters within fitness, and the relationship between growth rate and stability will provide insight into basic aspects of protein folding, function, and evolution. Theoreticians have long sought generalities that characterize the evolutionary process, and if such generalities exist, they should arise naturally from lower-level phenomena. The proposed experiments are designed to reveal such phenomena if they exist. Generalities about the evolution of protein thermal stability have not been forthcoming despite their relevance to a variety of fields, ranging from the evolution of extremophiles to basic questions about evolvability and the rational design of enzymes for industrial uses. Thermal stability is thought actually to promote evolvability by buffering against deleterious pleiotropic effects of mutations. The results will also aid in adjusting current models of adaptation to improve their realism and increase the accuracy of their predictions. Pleiotropy is a defining feature of Fisher's geometric model, and the proposed experiments will quantify the pleiotropic effects of mutations contributing to adaptation, providin information about the types of movements possible in multidimensional phenotypic space. They will also provide estimates of the main parameters in the mutational landscape model. Although epistasis has been widely documented, its molecular basis remains elusive. The results will provide a wealth of information about epistasis and its causes for beneficial mutations, about which few data exist. Many pathogens must survive under harsh conditions between infections and can potentially evolve greater virulence as a result. The proposed experimental system will serve as a model for studying the implications of this type of selection and provide insights into the evolution of infectious diseases.